Screening of Monoamine Oxidase Inhibitors from Seeds of Nigella glandulifera Freyn et Sint. by Ligand Fishing and Their Neuroprotective Activity

Nigella glandulifera is a traditional medicinal plant used to treat seizures, insomnia, and mental disorders among the Tibetan and Xinjiang people of China. Recent pharmacological research indicates that the seeds of this plant have a neuroprotective effect; however, the chemical components responsible for this effect are unknown. Monoamine oxidase B (MAO-B) has been recognized as a target for developing anti-Parkinson’s disease drugs. In this work, MAO-B functionalized magnetic nanoparticles were used to enrich the enzyme’s ligands in extracts of N. glandulifera seeds for rapid screening of MAO-B inhibitors coupled with HPLC-MS. Tauroside E and thymoquinone were found to inhibit the enzyme with IC50 values of 35.85 μM and 25.54 μM, respectively. Both compounds exhibited neuroprotective effects on 6-OHDA-induced PC-12 cells by increasing the cell viability to 52% and 58%, respectively, compared to 50% of the injured cells. Finally, molecular docking indicated strong interactions of both inhibitors with the enzyme. This work shows that MAO-B functionalized magnetic nanoparticles are effective for rapid screening of anti-PD inhibitors from complex herbal mixtures and, at the same time, shows the promising potential of this plant’s seeds in developing anti-PD drugs.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disorder and constitutes a societal health problem, especially among older people around the world [1]. PD affects motor and non-motor neurons and exhibits various symptoms such as muscle tremors, rigidity, bradykinesia, loss of stability, and body gaits [2]. Pathological characteristics of PD include the aggregation of alpha-synuclein in the intraneuronal cytoplasm, known as Lewy bodies, and the progressive damage of dopamine neurons in the substantia nigra pars compacta [3]. To date, there is no known cure for Parkinson's disease. The treatments for this disease, either medications or surgical procedures, are useful only for controlling the symptoms. Several influencing factors such as oxidative stress, mitochondrial dysfunction, genetics, age, environment, gender, and ethnicity have been highlighted as possible predisposing factors to Parkinson's disease and other related neuronal disorders [4,5]. Globally, there is an increasing number of people living with Parkinson's disease, as well as several other neurodegenerative diseases. For instance, in China, it is projected that PD patients may increase to approximately 5 million within a decade, thus accounting for a huge proportion of people over 60 years old [6][7][8]. Further, the cost of anti-PD medication exceeds the average person's economic capacity and creates a huge burden of care [9]. docking interaction were also investigated to evaluate the mechanisms of the interaction of the ligand with the identified MAO-B compounds.

Characterization of the Immobilized MAO-B
Fourier transform infrared (FT-IR) spectrometry was used to characterize the MNPs@SiO2, MNPs@COOH, and MNPs@MAO-B, as shown in Figure 1. The absorption peak at 629 cm −1 in Figure 1A corresponds to the stretching vibration of the Fe-O bond in the MNPs. The peaks for MNPs@COOH ( Figure 1B) at 1624 cm −1 and 1412 cm −1 were due to the N-H bending and the C-O stretching vibration, respectively, indicating that the amino and carboxyl groups were successfully modified on the MNPs@SiO2. In the spectrum of MNPS@MAO-B ( Figure 1C), the absorption peaks at 1641 cm −1 and 1050 cm −1 are stronger compared to those in MNPs@COOH, which indicates that there was a significant increase in the amount of amino, carboxyl, and peptide bonds in the structure of MAO-B [40]. The results imply that MAO-B was successfully immobilized on the MNP surface. Moreover, the amount of MAO-B immobilized on MNPS@COOH was found to be 48 μg/mg using the Coomassie Brilliant Blue stain method and Bovine Serum Albumin (BSA) as a standard.

Identification of the Ligands of MAO-B
The bioactive ligands bound as MNPs@MAO-B from S0 were dissociated with 500 μL of 50% acetonitrile-water (ACN) and denoted as S5. The S0 showed several peaks, while the S5 showed only two corresponding major ligands in the chromatogram ( Figure  2). Similar procedures and protocols have been successfully utilized on several MNP ligand fishing techniques, thus confirming the reliability of the magnetic nanoparticle solid phase extraction [41,42]. Furthermore, using MNPs@COOH as the blank control showed that no compound was observed in the corresponding HPLC-UV, confirming a non-specific binding on MNPs@MAO-B. The ligands were identified by HPLC-MS (Figures S1 and S2). The MS spectra revealed their molecular weights (Table S1). The molecular weight of ligand 1 was 750 (m/z 773, [M+Na] + ). Ligand 2 had a molecular weight of 166 (m/z 167, [M+H] + ). A comparison with the previously reported isolated compounds in the

Identification of the Ligands of MAO-B
The bioactive ligands bound as MNPs@MAO-B from S0 were dissociated with 500 µL of 50% acetonitrile-water (ACN) and denoted as S5. The S0 showed several peaks, while the S5 showed only two corresponding major ligands in the chromatogram ( Figure 2). Similar procedures and protocols have been successfully utilized on several MNP ligand fishing techniques, thus confirming the reliability of the magnetic nanoparticle solid phase extraction [41,42]. Furthermore, using MNPs@COOH as the blank control showed that no compound was observed in the corresponding HPLC-UV, confirming a non-specific binding on MNPs@MAO-B. The ligands were identified by HPLC-MS ( Figures S1 and S2). The MS spectra revealed their molecular weights (Table S1). The molecular weight of ligand 1 was 750 (m/z 773, [M+Na] + ). Ligand 2 had a molecular weight of 166 (m/z 167, [M+H] + ). A comparison with the previously reported isolated compounds in the literature revealed that ligand 1 was tauroside E, and ligand 2 was thymoquinone [32,43]. The chemical structures of the two ligands are shown in Figure 3.

MAO-B Ligands from N. glandulifera
The preliminary MAO-B inhibitory assay showed that the inhibitory rates of both ligands (0.05 mg/mL) were above 50%. The half-maximal inhibitory concentration (IC50) values for ligands 1 and 2 were calculated to be 35.85 ± 0.03 μM and 25.54 ± 0.05 μM, respectively (Table 1). This is the first report on the MAO-B inhibitory activity of both compounds while supporting the ethnomedicinal claims of N. glandulifera as an anti-neurodegenerative herb and a functional food. Furthermore, previous studies report that several compounds from herbal medicines, such as caffeic acid, catechin, and esculin, possess significant MAO-B inhibitory effects [44][45][46][47]. Our findings support the conclusion of previous studies that medicinal plants are a rich source for discovering inhibitors of MAO-B [46,47]. literature revealed that ligand 1 was tauroside E, and ligand 2 was thymoquinone [32,43]. The chemical structures of the two ligands are shown in Figure 3.

MAO-B Ligands from N. glandulifera
The preliminary MAO-B inhibitory assay showed that the inhibitory rates of both ligands (0.05 mg/mL) were above 50%. The half-maximal inhibitory concentration (IC50) values for ligands 1 and 2 were calculated to be 35.85 ± 0.03 μM and 25.54 ± 0.05 μM, respectively (Table 1). This is the first report on the MAO-B inhibitory activity of both compounds while supporting the ethnomedicinal claims of N. glandulifera as an anti-neurodegenerative herb and a functional food. Furthermore, previous studies report that several compounds from herbal medicines, such as caffeic acid, catechin, and esculin, possess significant MAO-B inhibitory effects [44][45][46][47]. Our findings support the conclusion of previous studies that medicinal plants are a rich source for discovering inhibitors of MAO-B [46,47].

MAO-B Ligands from N. glandulifera
The preliminary MAO-B inhibitory assay showed that the inhibitory rates of both ligands (0.05 mg/mL) were above 50%. The half-maximal inhibitory concentration (IC 50 ) values for ligands 1 and 2 were calculated to be 35.85 ± 0.03 µM and 25.54 ± 0.05 µM, respectively (Table 1). This is the first report on the MAO-B inhibitory activity of both compounds while supporting the ethnomedicinal claims of N. glandulifera as an antineurodegenerative herb and a functional food. Furthermore, previous studies report that several compounds from herbal medicines, such as caffeic acid, catechin, and esculin, possess significant MAO-B inhibitory effects [44][45][46][47]. Our findings support the conclusion of previous studies that medicinal plants are a rich source for discovering inhibitors of MAO-B [46,47].

Enzymatic Kinetic Study
The enzymatic kinetics of the enzyme inhibitors contribute additional information about the inhibitors' specificity and effectiveness for drug development [48]. The enzymatic kinetics of ligands 1 and 2 were assayed using Lineweaver-Burk plots which were plotted by the inverse of the substrate concentrations and velocities in the presence of varying concentrations of the MAO-B inhibitor (0 × IC 50 , 1/4 IC 50 , 2/4 IC 50 , 3/4 IC 50 , 4/4 IC 50 , and 5/4 IC 50 ). As shown in Figure 4A, six lines of Lineweaver-Burk plots of ligand 1 intersect the y-axis, revealing that ligand 1 is a competitive inhibitor. Competitive inhibitors compete with the substrate for the enzyme's active site. On the other hand, six straight lines of ligand 2 intersect the x-axis, as shown in Figure 4B, indicating that ligand 2 inhibits MAO-B in a non-competitive manner, which means that it binds to a site other than the active site of the enzyme.

Enzymatic Kinetic Study
The enzymatic kinetics of the enzyme inhibitors contribute additional information about the inhibitors' specificity and effectiveness for drug development [48]. The enzymatic kinetics of ligands 1 and 2 were assayed using Lineweaver-Burk plots which were plotted by the inverse of the substrate concentrations and velocities in the presence of varying concentrations of the MAO-B inhibitor (0 × IC50, 1/4 IC50, 2/4 IC50, 3/4 IC50, 4/4 IC50, and 5/4 IC50). As shown in Figure 4A, six lines of Lineweaver-Burk plots of ligand 1 intersect the Y-axis, revealing that ligand 1 is a competitive inhibitor. Competitive inhibitors compete with the substrate for the enzyme's active site. On the other hand, six straight lines of ligand 2 intersect the X-axis, as shown in Figure 4B, indicating that ligand 2 inhibits MAO-B in a non-competitive manner, which means that it binds to a site other than the active site of the enzyme.

Molecular Docking
Molecular docking is a computer-aided technology used to evaluate potential drug candidates and attribute features of the receptor and the interaction based on the theoretical role of the ligands [49]. The 2D and 3D molecular docking simulations of the ligands to the enzyme are shown in Figure 5. The lowest binding affinity energies of 1 and 2 were −9.3 kcal/mol and −7.2 kcal/mol, respectively, compared with safinamide (−11.79 kcal/mol) [30]. It was observed in this study that compound 2 showed better MAO-B inhibitory activity compared with compound 1, but the molecular docking investigation revealed that the affinity of compound 1 to the enzyme is stronger than that of compound 2. Specifically, compound 1 showed two main conventional hydrogen bonds at Glu-391 and Tyr-393. Furthermore, there are alkyl and π-alkyl interactions between Pro-277, His-252, Leu-250, Tyr-237, and Pro-234 of the enzymes to the sugar moieties of compound 1. As shown in Figure 5, compound 1 formed strong conventional hydrogen bonding with residues Glu-391 and Tyr-393, as well as alkyl and π-alkyl interactions with residues Pro-277, His-252, Leu-250, Tyr-237, and Pro-234. Similarly, compound 2 interacted with MAO-B by forming conventional hydrogen bonds with the residue of Tyr-435, as well as π-alkyl and alkyl

Molecular Docking
Molecular docking is a computer-aided technology used to evaluate potential drug candidates and attribute features of the receptor and the interaction based on the theoretical role of the ligands [49]. The 2D and 3D molecular docking simulations of the ligands to the enzyme are shown in Figure 5. The lowest binding affinity energies of 1 and 2 were −9.3 kcal/mol and −7.2 kcal/mol, respectively, compared with safinamide (−11.79 kcal/mol) [30]. It was observed in this study that compound 2 showed better MAO-B inhibitory activity compared with compound 1, but the molecular docking investigation revealed that the affinity of compound 1 to the enzyme is stronger than that of compound 2. Specifically, compound 1 showed two main conventional hydrogen bonds at Glu-391 and Tyr-393. Furthermore, there are alkyl and π-alkyl interactions between Pro-277, His-252, Leu-250, Tyr-237, and Pro-234 of the enzymes to the sugar moieties of compound 1. As shown in Figure 5

Neuroprotective Effect of the Inhibitors on 6-OHDA-Induced PC-12 Cells
The 6-OHDA neurotoxin is a widely utilized method of studying the therapeutic potential of drugs to treat PD. Since 6-OHDA shows the ability to cause the degeneration of dopaminergic neurons, and pheochromocytoma-12 (PC-12) cells injured by 6-OHDA showed a PD-like cellular model for the screening of anti-PD compounds, we assayed the neuroprotective effect of the identified inhibitors against 6-OHDA-induced PC-12 cells. The concentration of 6-OHDA was stabilized at 300 μM by comparing the damage degree to the cells caused by a series of concentrations (25 μM, 50 μM, and 100 μM). Compared to the PD-like model cells, the viability of cells pre-treated with ligand 1 at 25 μM increased from 50% to 58% and was statistically significant (p < 0.001); at 50 μM, it showed

Neuroprotective Effect of the Inhibitors on 6-OHDA-Induced PC-12 Cells
The 6-OHDA neurotoxin is a widely utilized method of studying the therapeutic potential of drugs to treat PD. Since 6-OHDA shows the ability to cause the degeneration of dopaminergic neurons, and pheochromocytoma-12 (PC-12) cells injured by 6-OHDA showed a PD-like cellular model for the screening of anti-PD compounds, we assayed the neuroprotective effect of the identified inhibitors against 6-OHDA-induced PC-12 cells. The concentration of 6-OHDA was stabilized at 300 µM by comparing the damage degree to the cells caused by a series of concentrations (25 µM, 50 µM, and 100 µM). Compared to the PD-like model cells, the viability of cells pre-treated with ligand 1 at 25 µM increased from 50% to 58% and was statistically significant (p < 0.001); at 50 µM, it showed a slightly protective effect of 52% (p < 0.01); and at 100 µM, there was a decrease (p < 0.05) in the neuroprotective effect. Notably, ligand 2 (50 µM) showed significantly (p < 0.001) increased neuroprotection compared with the model PC-12 cells from 50% to 55%, and the 100 µM concentration was 58% ( Figure 6). It was noticed that ligand 2 offered more neuroprotection than ligand 1 and that the higher the concentration, the stronger the neuroprotection. On the other hand, ligand 1 might have cell toxicity at high concentrations. These findings show that the two inhibitors offer a dose-dependent protective effect against 6-OHDA-induced PC-12 cells. The neuroprotective roles of some neuroactive components against 6-hydroxydopamine-induced PC-12 cells have previously been reported towards the development of Parkinson's disease drugs [40,50].
Plants 2023, 12, x FOR PEER REVIEW 7 of 13 a slightly protective effect of 52% (p < 0.01); and at 100 μM, there was a decrease (p < 0.05) in the neuroprotective effect. Notably, ligand 2 (50 μM) showed significantly (p < 0.001) increased neuroprotection compared with the model PC-12 cells from 50% to 55%, and the 100 μM concentration was 58% ( Figure 6). It was noticed that ligand 2 offered more neuroprotection than ligand 1 and that the higher the concentration, the stronger the neuroprotection. On the other hand, ligand 1 might have cell toxicity at high concentrations. These findings show that the two inhibitors offer a dose-dependent protective effect against 6-OHDA-induced PC-12 cells. The neuroprotective roles of some neuroactive components against 6-hydroxydopamine-induced PC-12 cells have previously been reported towards the development of Parkinson's disease drugs [40,50].

Materials, Reagents, and Instruments
Chemicals such as sodium hydroxide (NaOH), 3-aminopropyl-trimethoxysilane (APTMS), and dimethyl sulfoxide (DMSO) were obtained from the Chengdu Kelong chemical reagent factory (Chengdu, China). Monoamine oxidase B (MAO-B, 100.23 U/mL) was prepared in-house [12]. Kynuramine dihydrobromide was purchased from Sigma-Aldrich (St Louis, MO, USA). Safinamide mesylate and rasagiline were purchased from Meilunbio (Dalian, China). The MAO-B inhibition assay was carried out using Thermo Scientific Varioskan Flash equipped with a 96-well microplate (Thermo, Waltham, MA, USA). FT-IR spectra were recorded in KBr with a PerkinElmer FT-IR spectroscope (PerkinElmer, Waltham, MA, USA). Ultrapure water produced with a UP water purification (18.25 MΩ) system (Ultrapure, Chengdu, China) was used for HPLC. HPLC-grade methanol was obtained from JT Baker (Phillipsburg, NJ, USA). The HPLC system consisted of a Shimadzu LC-20 AD series equipped with a thermostatic column compartment, an SPD20A UV-vis detector (Shimadzu, Kyoto, Japan), and an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, 5 µm). a desolvation temperature of 350 • C; and a desolvation gas flow of 800 L/h, while the nebulizer was set at 0.8 Bar and the sample flow rate was set at 0.3 mL/min for the ESI-MS (Bruker Compass Data Analysis 4.0; microTOF-Q11-10203). For the FT-IR, the samples were pretreated with a tablet press using potassium bromide (KBr) in a dry environment, and the wavenumbers of the FT-IR measurement were set in the range of 450 to 4500 cm −1 .

Extraction of Seeds of N. glandulifera
N. glandulifera dried seeds were purchased from a local drug market in Lhasa, Tibet Autonomous Region of China. The whole plant, including the seeds, was identified by Professor Yong-Mei Zhang at the Chengdu Institute of Biology at the Chinese Academy of Sciences. A voucher specimen (2020-05) was deposited at the same institute. The seeds were pulverized and extracted with 80% aqueous MeOH under ultrasonication for 30 min. The organic solvent was removed to yield an aqueous solution, which was partitioned successively by petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to obtain the respective fractions.

Preparation and Characterization of MAO-B Functionalized Magnetic Nanoparticles
The MAO-B inhibitory activity of the two isolated ligands was tested according to the previous procedure with minor modifications [22]. In brief, 2.0271 g of FeCl 3 ·6H 2 O and 0.7407 g of FeCl 2 ·4H 2 O in 1:2 molar ratios were dissolved in 250 mL H 2 O, and ammonia water was added to adjust the pH to 9-10 before stirring under a nitrogen atmosphere for approximately 30 min at room temperature. An external magnet was used to separate the magnetic nanoparticles (MNPs), which were consecutively washed with water and ethanol. The MNPs were re-suspended in 150 mL of ethanol containing 400 µL of tetraethyl orthosilicate (TEOS), followed with the addition of ammonia water to adjust the pH to 9-10 and stirred for 5 h to produce a core-shell structure of MNPs@SiO 2 . The latter was separated using an external magnet, washed with water and ethanol, and then mixed with 2 mL of 3-aminopropyltriethoxysilane (APTMS) in 90 mL ethanol containing 1 mL water at 35 • C to obtain the amino-terminated MNPs (MNPs@NH 2 ). Then, 500 mg of lyophilized MNPs@NH 2 was treated with 3 g of succinic anhydride in 30 mL of dimethyl formamide (DMF) to terminate the MNPs with carboxyl groups as MNPs@COOH. Subsequently, 3 mg of MNPs@COOH was dispersed in 3 mL of MES buffer (PBS 50 mM, pH 7.4) containing 10 mM EDC·HCL and 20 mM NHS, and vortexed for 30 min, then MAO-B (2.5 U/mL) was added and incubated for 24 h at 25 • C. Finally, the obtained MNPs immobilizing the MAO-B (MNPs@MAO-B) were separated by a magnet and washed three times with PBS (50 mM, pH 7.4) to dissociate the unbound enzyme. To confirm the functionality of the synthesized magnetic nanoparticles, a Fourier transform infrared (FT-IR) spectrometer was used to characterize MNPs@SiO 2 , MNPs@COOH, and MNPs@MAO-B, as previously described in our laboratory.

Ligand Fishing of N. glandulifera
The n-BuOH fraction of the N. glandulifera extract was used for the ligand fishing test due to its moderate MAO-B inhibitory activity. First, the n-BuOH fraction was filtered with a 0.22 µm filtration membrane, concentrated to dryness, and diluted in PBS (50 mM, pH 7.4) to a concentration of 1 mg/mL, denoted as S0. A total of 3 mL of S0 was added to a 5 mL Eppendorf tube containing 20 mg of MNPs@MAO-B. The tube was oscillated for 30 min at room temperature, and the MNPs@MAO-B adsorbed with ligands of the enzyme were separated by an external magnet and washed three times using PBS (50 mM, pH 7.4). Then, 500 µL of 50% ACN was used to dissociate the ligands bound to the MNPs@MAO-B, denoted as S5. The S0 and S5 were analyzed by HPLC to determine the possible ligands of the enzyme.

Isolation of the Target Ligands
The two peaks that appeared in the high-performance liquid chromatography (HPLC) chromatogram of S5 were noted to be ligands of the enzyme. Under the guidance of HPLC, the n-BuOH fraction was subjected to column chromatography on ODS (MeOH/H 2 O, 20:80, 50:50, and 100:0, v/v), silica gel (CH 2 Cl 2 /MeOH, from 100:1 to 1:1, v/v), and Sephadex LH-20 (MeOH 100%, v/v) to afford the two compounds ( Figure S3).

Monoamine Oxidase B Inhibition Assay
The MAO-B inhibitory activity of the two isolated ligands was tested according to the previous procedure with minor modifications [21]. First, 50 µL of MAO-B (2.5 U/mL) was incubated with 100 µL of the test compound (inhibitor) in a 96-well ELISA plate at 37 • C for 10 min. Secondly, 50 µL of kynuramine (50 µM) was added to incubate at 37 • C for 30 min before 80 µL of NaOH (2N) was introduced to end the reaction. The absorbance was read using a multimode microplate reader (Varioskan Flash, 310/400 ňex/em). Meanwhile, 0.01 mg/mL of safinamide, a known MAO-B inhibitor for the treatment of PD, was used as a positive control. Assays were performed in triplicates. Data were analyzed using GraphPad Prism 6.0 software and expressed as the mean ± standard deviation. The percentage inhibition and the half-maximal inhibitory (IC 50 ) values of the active compounds for MAO-B were determined using Ellman's method [51]: where B0 represents the absorbances of the test blank (PBS and MAO-B), B1 represents the absorbances of the sample, and B2 represents the absorbances of the control blank (PBS, MAO-B, and Kynuramine).

Enzymatic Kinetic Study
The Lineweaver-Burk plot was used for the kinetic assay, the concentrations of kynuramine were between 20 and 180 µM, and the different folds of the ligands were prepared based on the IC 50 . The concentration of the MAO-B used was 2.5 U/mL, and the reaction was monitored using a plate reader after 10 min.
The Lineaweaver-Burk plot was calculated as: where [S] is the concentration of MAO-B, and v and V max represent the enzyme reaction rate and the maximum enzymatic reaction velocity, respectively.

Molecular Docking
Molecular docking was optimized to predict the optimal binding mode of the ligandreceptor complex by studying the binding affinity and the amino acid residue environments [52]. The MAO-B protein (PDB ID: 2V5Z) was retrieved from (http://www.rcsb. org/pdb/accessed on 10 September 2022) and the three-dimensional (3D) structure of MAO-B (Homo sapiens) was downloaded from the Protein Data Bank (http://www.rcsb. org/pdb/home/home.do accessed on 10 September 2022). The ligands were drawn with ChemBio-Draw Ultra 14.0, while the protein and compounds were converted to PDBQT (Protein Data Bank, Partial Charge (Q) and Atom Type (T)) files, and the docking input files were generated using AutoDock Tools 1.5.6 (Scripps Research, 211 San Diego, CA, USA). The genetic algorithm (GA) was set up 1000 times to run the hit, and the value of exhaustiveness was set to 27. The search grid was set up at center x = 50.757, y = 155.963, and z = 27.636, and the dimension sizes were set up at x = 31.58, size y = 35.50, and size z = 30.066. Gasteiger charges and the number of torsions were set for metabolites, and polar hydrogens were merged. For the receptor polar hydrogens, Kollman charges were added, solvation parameters were assigned by default, and for Vina docking, the default parameters were used. The binding energy predicted by AutoDock Vina with the lowest energy mode was selected as the best binding energy affinity and the output files were visualized using Discovery Studio and PyMol.
3.9. Protective Effect of the Ligands on 6-OHDA-Induced PC-12 The PC-12 cells were obtained from Hunan Fenghui Biotechnology Co., Ltd. (Changsha, China). The 6-hydroxydopamine (6-OHDA) was used because it is a toxic oxidative metabolite of dopamine and has been used effectively to evaluate potential anti-Parkinsonism agents [53]. The cells were cultured in DMEM supplemented with 5% FBS and 1% penicillin-streptomycin, and maintained at 37 • C with 5% CO 2 . The cells were trypsinized when they achieved over 90% growth on the bottom and seeded in 96-well microplates (1 × 10 4 cells/well) to incubate in the same medium and environment as above. After being cultured for 24 h, PC-12 cells were treated with 100 µL of each test compound at various concentrations (25 µM, 50 µM and 100 µM) for 2 h, and then the cells were incubated with 6-OHDA for another 24 h. At the same time, rasagiline was used as the positive control in this experiment because safinamide failed to exhibit significant protective effects on PC-12 cells as rasagiline. The optical densities were measured at 450 nm using a multimode microplate reader (Thermo Fisher, Waltham, MA, USA).

Data Analysis
Data were expressed as the mean ± standard deviation (SD) from three replicate experiments. GraphPad Prism 6.0 was used as the statistical analysis software. The oneway analysis of variance (ANOVA) and the mean ± standard deviation (n = 3) were separated. The p < 0.05 was considered to be statistically significant. Finally, ChemDraw version 14 (student version) was used to draw the chemical structures in this study.

Conclusions
This study identified two MAO-B inhibitors (tauroside E and thymoquinone) from N. glandulifera dried seeds using a ligand fishing technique based on MAO-B functionalized magnetic nanoparticles. In this work, the in vitro MAO-B inhibition activity and neuroprotective potential on 6-OHDA-induced PC-12 cells of both ligands were reported for the first time, with IC 50 values of 35.85 µM and 25.54 µM, respectively. Both compounds exhibited neuroprotective effects on 6-OHDA-induced PC-12 cells by increasing viability by 52% and 58%, respectively. The molecular docking interactions revealed that these inhibitors might play significant roles in neuroactive ligand-receptor interactions in the major neurodegenerative pathway and PD. The findings contribute to the fast screening of neuroprotective components of N. glandulifera using magnetic nanoparticle ligand fishing, and justify its health benefits as a food and an herb in managing PD and other neurological diseases.
Author Contributions: Conceptualization, X.L. and C.M.; formal analysis, E.A.A. and C.M.; investigation, E.A.A. and Y.H.; writing-original draft preparation, E.A.A. and Y.Z.; writing-review and editing, E.A.A. and X.L.; visualization, E.A.A. and X.B.; supervision, X.L.; project administration, Y.Z. and X.B.; funding acquisition, Y.Z. and X.L. All authors have read and agreed to the published version of the manuscript.

Funding:
We acknowledge the financial support from Sichuan Science and Technology Program (2022ZYD0032 to X.L.) and the National Science and Technology Resource Sharing Service Platform project of the Ministry of Science and Technology of China 508 (E0117G1001 to Y.Z.). We also thank the CAS-TWAS President fellowship for student support (to E.A.A.).

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.